The primary pathway by which telomeres are maintained in most species relies on the enzyme telomerase. In model organisms such as yeast as well as in human cells, continual replenishment of chromosome termini by telomerase is critical in order to ensure continuous cellular proliferation. The contribution of telomerase to human health, with regard to both cancer biology and human aging, is substantial. Telomerase activity is up- regulated in ~90% of human cancers. Conversely, genetically inherited reductions in telomerase function have a significant impact on age-related phenomena, encompassing a broad spectrum of telomere-mediated diseases referred to as syndromes of telomere shortening. However, despite the importance of telomerase, a unified model to explain how telomerase is regulated in vivo to maintain telomere length homeostasis is still lacking. This deficit stems at least in part from limitations of the current experimental systems that are widely used for telomerase studies. We have addressed some of the prior obstacles to telomerase analysis in S. cerevisiae, through the development of several biochemical assays as well as a novel genetic strategy that can target surface residues even in the absence of structure. In our first Specific Aim, we propose an integrated analysis of the yeast telomerase holoenzyme complex in which telomerase genetics, biochemistry and enzymology can be assessed in one experimental system. The proposed experiments are designed to identify new telomerase-interacting regulatory factors, determine how catalytic features of the telomerase enzyme impact telomere length in vivo, and examine how telomerase assembly and disassembly through the cell cycle influences telomere homeostasis. This analysis will be aided by a comprehensive panel of genetic reagents in the three subunits of telomerase. The goal is to provide a temporal and enzymatic understanding of how elongation of individual telomeres by telomerase is regulated in order to achieve telomere homeostasis in wild type cells. An understanding of telomerase regulation provides only an incomplete picture of the contribution of telomere biology to cellular proliferation, however. In the absence of telomerase, telomere attrition results in an eventual block to further cell division, referred to as replicative senescence. The development of a semi- quantitative assay for senescence that measures even slight differences in proliferation has demonstrated that proliferation of telomerase-defective yeast even during early stages of replicative senescence is controlled by multiple pathways. This points to a largely unexplored network that is likely to be as complex as the genetic interactions that regulate telomere length in the presence of telomerase. The goal of Specific Aim 2 is to define this genetic network, using a rapid quantitative assay designed to monitor cell proliferation in the absence of telomerase, combined with a single-nucleotide-resolution assay that monitors how the patterns of telomere erosion change in response to defects in different pathways.